Every biochemical process that happens in a eukaryotic cell relies upon a molecular information flow that leads from receptors that inform the cell about its environment all the way to the molecular effectors that determine the appropriate cellular response. A proper information transmission requires a high degree of organization where the molecular players are organized into different cellular compartments so that the specificity of the cellular response can be properly maintained. Breakdown of this organization is the ultimate cause of all human diseases even if the affected molecular pathways differ according to the kind of disease, such as cancer, diabetes or neurodegenerative diseases just to name a few. Research described in this report has focused on the question of how cells organize their internal membranes to provide a structural framework on which molecular signaling complexes assemble to ensure proper information processing. These cellular processes are often targeted by cellular pathogens such as viruses to force the cells to produce the pathogen instead of performing the cells normal functions. Better understanding of these processes not only can provide new strategies to fight various human diseases but also to intercept the life cycle of cellular pathogens offering an alternative to antimicrobial drugs. Inositol phospholipids are a class of phospholipids that are present in tiny amounts but have very important regulatory functions in the they organize protein signaling complexes on specific membrane compartments. They are produced by phosphoinositide kinases that can phosphorylate specifically one of three positions of the inositol ring of phosphatidylinositol (PI). In this review period we studied the role of phosphatidylinositol 4-kinase alpha (PI4K2A) in autophagosome-lysosome fusion. Autophagy is an important cellular process that helps clear damaged organelles and also allows cells to recycle useful nutrients from degraded organelles during starvation. In earlier studies we found that PI4K2A interacted with the GABARAP protein, one of the family of proteins that is important for autophagy. This observation was confirmed in a subsequent study by the Albanesi group, which also showed that PI4K2A was important for the acidification of autophagic vesicles. Therefore, we have generated HEK293 cells with inactivated PI4K2A using CRISPR/Cas9 gene editing and studied their properties including those important for autophagy. We found increased number of LC3 (a widely used marker of autophagy) positive vesicles that failed to acidify in the PI4K2A knockout (K/O) cells. We determined the distribution of phosphatidylinositol 4-phosphate (PI4P) along the endosomal network through the introduction of a novel bioluminescence resonance energy transfer (BRET)-based assay that allowed for the quantification of levels of PI4P within various Rab positive endosomes. This analysis showed that the levels of PI4P are highest in Rab7 positive endosomes and are kept very low in Rab4, Rab5 and Rab11 compartments. PI4K2A was responsible for 80% of the PI4P produced in the Rab7 compartment, with the rest produced by PI4K2B. Conversion of PI4P to PI(4,5)P2 caused the inactivation of Rab7 and release of PLEKHM1, an important adapter protein linking autophagic vesicles with lysosomes, from the Rab7 compartment. We also found that PI4P in the Rab7 compartment was converted to PI(4,5)P2 by endogenous PIP5Kgamma in wild-type, but not in PI4K2A K/O cells and that PIP5Kgamma knock-down in parental HEK293 reproduced the effects of PI4K2A inactivation by inhibiting autophagic vesicle acidification. These data suggested a sequence of events by which PI(4,5)P2 generation from the PI4P produced by PI4K2A in Rab7 positive endosomes caused Rab7 inactivation, presumably by activating one or more Rab7 GTPase activating (GAP) protein(s). This chain of events contributes to the cycling of some Rab7 effectors, including PLEKHM1, which is necessary for the fusion of lysosomes with autophagosomes. The importance of these studies is that it revealed a hitherto unknown role of PI4P to PI(4,5)P2 conversion in the control of Rab7 activation state and serving as a regulator of trafficking decisions in the endocytic pathway. In a collaborative project with the group of Drs Sue Goo Rhee and Dongmin Kang in the Ewha Womans University of Seoul, Republic of Korea, the effects of reactive oxygen radicals were examined on the Sac1 phosphatase enzyme. Sac1 dephosphorylates PI4P in the endoplasmic reticulum (ER) and plays crucial roles in the control of non-vesicular lipid transport driven by PI4P gradients between various organelles and the ER. We found that hydrogen peroxide (H2O2) has a profound effect on the cellular level of PI4P, which was most prominent in the Golgi and Rab7-positive endosomes, but also manifested in PI4P increase in the plasma membrane (PM). Upon H2O2 exposure, Sac1undergoes reversible inactivation in mammalian cells due to the oxidation of its catalytic Cys389 residue, which then forms an intramolecular disulfide with Cys392. The Korean group has also shown that this oxidation process also takes place during stimulation of cells by EGF and that Duox enzymes are responsible for the endogenous H2O2 production under these conditions. These findings revealed an important regulation of the Sac1 phosphatase by reversible oxidation, thereby controlling both the signaling function of PI4P and its effectiveness of driving lipid transport at membrane contact sites. In light of the pivotal role of PI4P in virus replication and the role of type II PI4Ks in endosomal functions, we wanted to identify small molecule inhibitors for the PI4K2A enzyme. To date, pharmaceutical companies have focused exclusively on inhibitors for type III PI4Ks, such as PI4KA and PI4KB, but no efforts have been devoted by Pharma to identify inhibitors of type II PI4Ks. We have developed a PI4K activity assay for a small format suitable for high throughput screening (HTS) using a bacterially expressed human PI4K2A enzyme produced by Dr. Bouras group in Prague, Czech Republic. A high throughput screening was then performed by NCATS with 400,000 compounds from small molecule diversity collections. Sytravon, NPC & MLPCN collections were screened at top two doses (76 & 15 M final concentration). Based on the screening results, we identified 580 compounds with > 50% inhibitory activity. These compounds were re-tested at 7 doses in 1:3 serial dilution to confirm their activity. Further, they were counter-screened with ADP-Glo reagents in the absence of the enzyme to exclude artificially luminescent compounds, and with PI4KB, another structurally unrelated PI4K. The list was further narrowed by eliminating compounds that were known inhibitors of protein kinases or were deemed structurally unsuitable for further development, yielding 14 inhibitors shortlisted for further studies. These 14 inhibitors were then tested in a cellular assay using the BRET method to monitor PI4P levels in Rab7 endosomes that require PI4K2A. Two inhibitors were then found to have inhibitory effect on PI4K2A in the cell with an IC50 of 30 M. These two lead compounds (code named NC03 and NC02) were then modified by NCATS (NC03) and also by our medicinal chemist collaborator (Radim Nencka, Prague, Czech Republic, for NC02) to increase the potency and cellular availability. Unfortunately, these significant efforts have not yielded any improvement over the lead compounds. Since then a new screen has been performed and testing is under way to improve the newly identified lead compounds.

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Alvarez-Prats, Alejandro; Bjelobaba, Ivana; Aldworth, Zane et al. (2018) Schwann-Cell-Specific Deletion of Phosphatidylinositol 4-Kinase Alpha Causes Aberrant Myelination. Cell Rep 23:2881-2890
Balla, Tamas (2018) Ca2+ and lipid signals hold hands at endoplasmic reticulum-plasma membrane contact sites. J Physiol 596:2709-2716
Sohn, Mira; Korzeniowski, Marek; Zewe, James P et al. (2018) PI(4,5)P2 controls plasma membrane PI4P and PS levels via ORP5/8 recruitment to ER-PM contact sites. J Cell Biol 217:1797-1813
Várnai, Péter; Gulyás, Gerg?; Tóth, Dániel J et al. (2017) Quantifying lipid changes in various membrane compartments using lipid binding protein domains. Cell Calcium 64:72-82
Gulyás, Gerg?; Radvánszki, Glória; Matuska, Rita et al. (2017) Plasma membrane phosphatidylinositol 4-phosphate and 4,5-bisphosphate determine the distribution and function of K-Ras4B but not H-Ras proteins. J Biol Chem 292:18862-18877
Korzeniowski, Marek K; Wisniewski, Eva; Baird, Barbara et al. (2017) Molecular anatomy of the early events in STIM1 activation - oligomerization or conformational change? J Cell Sci 130:2821-2832
Levin, Roni; Hammond, Gerald R V; Balla, Tamas et al. (2017) Multiphasic dynamics of phosphatidylinositol 4-phosphate during phagocytosis. Mol Biol Cell 28:128-140
Kim, Yeun Ju; Guzman-Hernandez, Maria Luisa; Wisniewski, Eva et al. (2016) Phosphatidylinositol and phosphatidic acid transport between the ER and plasma membrane during PLC activation requires the Nir2 protein. Biochem Soc Trans 44:197-201
Egervari, Kristof; Potter, Gael; Guzman-Hernandez, Maria Luisa et al. (2016) Astrocytes spatially restrict VEGF signaling by polarized secretion and incorporation of VEGF into the actively assembling extracellular matrix. Glia 64:440-56
Klima, Martin; Tóth, Dániel J; Hexnerova, Rozalie et al. (2016) Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein. Sci Rep 6:23641

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